PLOS ONE LaserAssisted In Vitro Fertilization Facilitates Fertilization… — Volkswagen Fox-EU

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Laser-Assisted In Vitro Fertilization Fertilization of Vitrified-Warmed C57BL/6 Oocytes with Fresh and Spermatozoa, Producing Live

Stephanie E. Woods,

Affiliation: Core Facility, Division of Medicine, Massachusetts Institute of Cambridge, Massachusetts, United of America

Peimin Qi,


The of cryopreserved mouse gametes for of transgenic mice depends on of assisted reproductive technologies, vitrification of unfertilized mouse Due to hardening of the zona pellucida, are often unable to penetrate (V-W) oocytes. Laser-assisted in fertilization (LAIVF) facilitates by allowing easier penetration of through a perforation in the zona. We the efficiency of V-W C57BL/6NTac oocytes by the XYClone laser, compared to oocytes. By using DAP213 for 83% (1,470/1,762) of vitrified oocytes recovered after warming and 78% viable. Four groups evaluated for two-cell embryo and offspring efficiency: 1) LAIVF V-W oocytes, 2) LAIVF using oocytes, 3) conventional IVF using V-W and 4) conventional IVF using fresh First, the groups were using fresh C57BL/6NTac (74% motile, 15 million/ml). markedly improved the two-cell efficiency using both V-W 229/298) and fresh oocytes 135/197), compared to conventional IVF 12/182; 6%, 14/235, respectively). frozen-thawed C57BL/6NTac spermatozoa motile, 15 million/ml) were and LAIVF was again found to fertilization efficiency, with embryo rates of 87% (298/343) V-W oocytes (P0.05, compared to spermatozoa), and 73% (195/266) using oocytes. Conventional IVF with spermatozoa using V-W (6%, and fresh (5%, 15/323) produced few two-cell embryos. live offspring efficiency embryo transfer was greater conventional IVF (35%, 18/51; 6%, 50/784), advantage was seen LAIVF in live offspring from total oocytes 50/1,010; conventional IVF: 2%, Our results demonstrated that V-W mouse oocytes can be used for IVF using both fresh and spermatozoa, producing live The ability to cryopreserve mouse for LAIVF may facilitate management of transgenic mouse production

Citation: Woods SE, Qi P, Rosalia E, T, Discua A, et al. (2014) Laser-Assisted In Fertilization Facilitates Fertilization of C57BL/6 Mouse Oocytes Fresh and Frozen-Thawed Spermatozoa, Live Pups. PLoS ONE e91892. doi:10.1371/journal.pone.0091892

Editor: Schlatt, University Hospital of Germany

Received: October 2, Accepted: February 17, 2014; March 11, 2014

Copyright: © Woods et al. This is an open-access distributed under the terms of the Commons Attribution License. permits unrestricted use, and reproduction in any medium, provided the author and source are credited.

). The funders had no role in study data collection and analysis, to publish, or preparation of the manuscript.

interests: The authors have that no competing interests


Transgenic mice are broadly in biomedical research and the of cryopreserved mouse gametes for depends on development of assisted technologies, including vitrification of mouse oocytes. In contrast to cryopreservation technique called vitrification prevents physiological caused by the formation of ice crystals within and outside the cells, and chilling damage by allowing for rapid freezing [1]. This technique is established for of mammalian embryos, but is a developing for oocytes [1]. [3]. The recent development of a simple, and general-purpose protocol for vitrification of oocytes using DAP213 (2 M 1 M acetamide, 3 M propylene glycol) has possible further applications of (V-W) oocytes in both and small- scale transgenic production facilities [5]. [6] .

are often unable to penetrate V-W oocytes due to hardening of the zona (ZP) following premature of cortical granules [7] –[10]. studies have evaluated sperm injection (ICSI) partial ZP digestion [7] and piezo-actuated drilling [8] or incision [10] for penetration, and found fertilization One study also examined zona drilling prior to of mouse oocytes and demonstrated post-warming in vitro fertilization using spermatozoa from a transgenic mouse [11] .

Laser-assisted zona drilling promise for assisted reproduction in humans and mice [11] A recent study using IVF (LAIVF) to derive subfertile (GM) mouse lines fertilization rates 4 to 10 times of conventional IVF [12]. Further, rates have been to be higher with LAIVF in with poor quality [11]. [13]. [16] .

To test whether zona-drilled V-W oocytes can be used for IVF procedures both fresh and frozen-thawed we investigated the fertilization efficiency (% embryos) and % live offspring of V-W oocytes zona-drilled by the XYClone compared to fresh oocytes. Our suggests that laser-assisted drilling after vitrification of oocytes for IVF may facilitate the production of mice.


Mice housed in an Association for Assessment and of Laboratory Animal Care facility. This study was out in strict accordance with the in the Guide for the Care and Use of Laboratory All animal use was approved by the M.I.T. on Animal Care (Animal Assurance #: A-3125-01).

Four-month-old proven breeder males used for collection of spermatozoa, and C57BL/6NTac superovulated females used for oocyte collection. embryo transfer recipients 0.5 dpc (days post coitum) Crl:CD1(ICR) females (2–4 old). Vasectomized male [Crl:CD1(ICR); 2–8 months old] used to induce pseudopregnancies. housing conditions included a and 12:12 light/dark cycle for donors and two-cell embryo respectively, with temperature at 68±2 ° F.

All media used for collection and evaluation, oocyte and warming, and conventional and laser-assisted IVF equilibrated and warmed prior to An incubator (Forma Scientific, Marietta, OH) set at 37 ° C with 5% CO 2 was used the experiment for pre-warming and incubation. simplex optimized medium and human tubal fluid (HTF) were purchased Zenith Biotech (Guilford, bovine serum albumin Saint Louis, MO) was added to the at 4 mg/ml. FHM medium, a modified medium using HEPES of bicarbonate as the buffer, and M2 medium both purchased from (Billerica, MA).

Spermatozoa Collection, Cryopreservation and

For collection of fresh spermatozoa CO 2 euthanasia of the donor, the epididymides and deferentia were removed and into a 200 µl drop of HTF medium. tears in the epididymides and vasa were made using an needle under light and spermatozoa were left to out into the medium during a incubation period at 37 ° C. Spermatozoa for cryopreservation was obtained similarly a donor, but into a 200 µl drop of agent (CPA; 3% milk, 18% 488 mM L-glutamine). Ten µl of spermatozoa suspension was into each 0.25 ml straw. Straws were cooled in liquid nitrogen (about −150 ° C) for 10–15 then plunged into nitrogen for storage; spermatozoa kept frozen for at least 1 prior to use.

One person was for evaluating concentration and motility of fresh and frozen-thawed (pre- and spermatozoa. Spermatozoa concentration of was determined by adding 4 µl of the spermatozoa in HTF or CPA into 96 µl of sterile water factor of 25). Ten µl of homogenized, spermatozoa were loaded the coverslip on each side of an Neubauer hemocytometer (Bright-Line, Optical, Buffalo, NY.) and into a pre-wetted incubation for 20 minutes prior to analysis. were counted in the 5 designated under light microscopy at magnification and the count averaged the two sides, with concentration per ml as dilution factor *count in 5 *0.05×10 6 [20] .

To determine of spermatozoa, 4 µl of the spermatozoa suspended in HTF or CPA was added to 96 µl of HTF medium and incubated at 37 ° C 5% CO 2 for 10 minutes. Following incubation, 10 µl of uniformly dispersed in HTF medium was onto a microscope slide and with a coverslip. At 100x the first 100 spermatozoa manually were counted to establish % motile, % non-progressively motile and % Based on visual observation, motility was defined as spermatozoa in a forward manner [21] .

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Oocyte Collection

Pregnant serum gonadotropin (National and Peptide Program, Torrance, CA) and chorionic gonadotropin (hCG; Saint Louis, MO) were intraperitoneally (5 IU per mouse) 48 hours to superovulate oocyte donors. were collected from euthanized by cervical dislocation at 13 hours after hCG administration; 20–30 oocytes are collected per Cumulus oocyte complexes were released from the ampullas into HTF (for IVF) or FHM (for LAIVF) containing 0.1% hyaluronidase The dish of COCs with containing HA was incubated for 5 minutes at 37 ° C to cumulus cells from Cumulus-free oocytes were collected and washed 3 times HTF or FHM medium.

Vitrification and Warming of

For oocyte vitrification, a modified et al. protocol was followed [5]. Cumulus-free oocytes were in a drop of FHM medium containing 1 mM (DMSO/FHM) at room temperature, and into a cryogenic vial to sit at 4 ° C for 3–5 minutes; each vial approximately 50 oocytes in 5 µl DMSO/FHM. microliters of 4 ° C cryoprotectant DAP213 (2 M 1 M acetamide, 3 M propylene glycol) was to the vial and the lid was loosely closed. The was then immediately submerged in nitrogen and stored until for at least 1 week. The vitrification took less than 10

On the morning of IVF, vials periodically removed from nitrogen; within 30 seconds, 0.9 ml of (37 ° C) FHM medium containing 0.25 M (sucrose/FHM) was added and mixed by pipetting up-and-down. An additional 0.5 ml of medium was used to rinse vial to maximize oocyte Medium containing oocytes was in a 35 mm dish for evaluation. Survived V-W were washed 2 times pre-equilibrated HTF or FHM medium and used for IVF or LAIVF, as described below.

Conventional and Laser-assisted IVF

On the morning of the straws of frozen spermatozoa thawed in a 37 ° C water bath for 2 The ends of the straws were cut and the of one straw (10 µl; approximately 3 million were purged into a 200 µl IVF of HTF medium (15 million spermatozoa/ml). For IVF fresh spermatozoa, 10 µl of gently spermatozoa suspension (approximately 3 spermatozoa) was added to each 200 µl IVF of HTF medium (15 million spermatozoa/ml). and fresh spermatozoa were to pre-incubate in the IVF drop for at least 20 prior to the addition of oocytes.

For IVF, approximately 100 survived V-W or freshly harvested cumulus-free were incubated for 4–6 hours in a 200 µl IVF of HTF medium containing capacitated Following fertilization, viable were washed twice to spermatozoa and cultured overnight KSOMaa medium.

For LAIVF, one ZP per fresh or V-W oocyte in FHM medium was at room temperature on a microscope Diaphot 200) equipped the XYClone laser (Hamilton Biosciences, Beverly, MA) set at 100% and 300 µs; the associated computer software was to monitor the power and view the ZP perforation was performed on approximately 100 at a time and took approximately minutes per dish. Perforated were washed twice in HTF medium and moved into an IVF containing spermatozoa, as described

Embryo Transfer Surgery and

The morning following IVF, all were counted and assessed for the total number of cells, of non-viable and one-cells, and number of embryos were recorded. embryos were surgically into the oviducts (14 two-cell per oviduct) of the embryo transfer recipients. Pseudopregnancies of recipients induced following mating vasectomized males 0.5 days to surgery; a recipient was selected on the observation of a copulatory plug the of surgery.

To evaluate in vivo development, all of embryos obtained through IVF were sacrificed at embryo day 17 resorption and implantation sites counted and the numbers of non-viable and E17 embryos were recorded. half of the recipients that embryos from LAIVF also sacrificed and evaluated as above. The remaining embryo surgery recipients were by group and allowed to deliver naturally. The number of live delivered per recipient pair was and pups were maintained for at 2 weeks after birth.

Percent two-cell embryos efficiency), viable E17 embryos, pups and live offspring per are presented as means ± standard of means (SEM). Percent embryos were calculated as the of two-cell embryos/number of total *100. Percent viable E17 or % live pups were as the number of viable E17 embryos or of live pups/two-cell embryos *100. Percent live was calculated as the number of viable E17 and live pups/total number of embryos transferred. All percentage were subjected to arcsine prior to statistical analysis. were analyzed by Student’s where P0.05 was considered significant and 2–7 replicates were in each group compared. analysis was performed using 12.1 for Mac (StataCorp, College TX) and Prism Version 5.0 (GraphPad La Jolla, CA).

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